Does freezing actually “freeze” information over time? Biomass conservation impact on metagenomic profiling of microbial communities from wastewater treating systems

Immediate DNA extraction from samples upon collection is not always feasible. Despite preservation efforts, sample storage can nonetheless distort metagenomic-based inferences. Numerous studies have investigated the storage impact on community profiling, yet no clear consensus has been reached. Accordingly, this paper evaluated the storage-induced biases in 16S rRNA amplicon sequencing of communities from activated sludge flocs and aerobic granular sludge reactors used for wastewater treatment. Different storage temperatures (RT, 4 °C, −20 °C, −80 °C) and durations (up to six months, with few samples extending to three years), as well as the use of glycerol as cryo-protectant, were evaluated. Briefly, thawing primarily affected the most abundant taxa but detectable losses were also observed in less abundant yet relevant taxa. Nevertheless, overall community profiles generally remained consistent enough to prevent sample misidentification, even under the least favourable conditions. Finally, glycerol addition resulted beneficial at −80 °C but not at −20 °C.